Fusion protein delivery system and uses thereof

ABSTRACT

The present invention provides a composition of matter, comprising: DNA encoding a viral Vpx protein fused to DNA encoding a protein. In another embodiment of the present invention, there is provided a composition of matter, comprising: DNA encoding a viral Vpr protein fused to DNA encoding a protein. The present invention further provides DNA, vectors and methods for expressing a lentiviral pol gene in trans, independent of the lentiviral gag-pol. A gene transduction element is optionally delivered to a lentiviral vector according to the present invention. Also provided are various methods of delivering a virus inhibitory molecule to a target in an animal. Further provided is a pharmaceutical composition.

RELATED APPLICATION

[0001] This patent application is a divisional of patent applicationSer. No. 08/947,516 filed Sept. 29, 1997, which is a file-wrappercontinuation of patent application Ser. No. 081421,982 filed Apr. 14,1995, of which both are incorporated herein by reference.

Background of the Invention

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields ofmolecular virology and protein chemistry. More specifically, the presentinvention relates to the use of Human and Simian Immunodeficiency Virus(HIV/SIV) Vpx and Vpr proteins, or amino acid residues that mediatetheir packaging, as vehicles for delivery of proteins/peptides tovirions or virus-like particles and uses thereof.

[0004] 2. Description of the Related Art

[0005] Unlike simple retroviruses, human and simian immunodeficiencyviruses (HIV/SIV) encode proteins in addition to Gag, Pol, and Env thatare packaged into virus particles. These include the Vpr protein,present in all primate lentiviruses, and the Vpx protein, which isunique to the HIV-2/SIV_(SM)/SIV_(MAC) group of viruses. Since Vpr andVpx are present in infectious virions, they have long been thought toplay important roles early in the virus life cycle. Indeed, recentstudies of HIV-1 have shown that Vpr has nucleophilic properties andthat it facilitates, together with the matrix protein, nuclear transportof the viral preintegration complex in nondividing cells, such as themacrophage. Similarly, Vpx-deficient HIV-2 has been shown to exhibitdelayed replication kinetics and to require 2-3 orders of magnitude morevirus to produce and maintain aproductive infection in peripheral bloodmononuclear cells. Thus, both accessory proteins appear to be importantfor efficient replication and spread of HIV/SIV in primary target cells.

[0006] Incorporation of foreign proteins into retrovirus particles haspreviously been reported by fusion with gag. Using the yeastretrotransposon Tyl as a retrovirus assembly model, Natsoulis and Boeketested this approach as a novel means to interfere with viralreplication. More recently, the expression of a murine retroviruscapsid-staphylococcal nuclease fusion protein was found to inhibitmurine leukemia virus replication in tissue culture cells.

[0007] The prior art lacks effective means of delivering or targetingforeign, e.g., toxic proteins to virions. The present invention fulfillsthis longstanding need and desire in the art.

SUMMARY OF THE INVENTION

[0008] Vpr and Vpx packaging is mediated by the Gag precursor and thusmust play an important role in HIV assembly processes. The presentinvention shows that Vpr and Vpx can be used as vehicles to targetforeign proteins to HIV/SIV virions. Vpr1 and Vpx2 gene fusions wereconstructed with bacterial staphylococcal nuclease (SN) andchloramphenicol acetyl transferase (CAT) genes. Unlike Gag or Polproteins, Vpr and Vpx are dispensable for viral replication inimmortalized T-cell lines. Thus, structural alteration of theseaccessory proteins may be more readily tolerated than similar changes inGag or Gag/Pol. Fusion proteins containing a Vpx or Vpr moiety should bepackaged into HIV particles by expression in trans, since theirincorporation should be mediated by the same interactions with Gag thatfacilitates wild-type Vpr and Vpx protein packaging.

[0009] Vpr and Vpx fusion proteins were constructed and their abilitiesto package into HIV particles were demonstrated. Fusion partnersselected for demonstration were: staphylococcal nuclease because of itspotential to degrade viral nucleic acid upon packaging and thechioramphenicol acetyl transferase because of its utility as afunctional marker. To control for cytotoxicity, an enzymaticallyinactive nuclease mutant (SN*), derived from SN by site-directedmutagenesis was also used. This SN* mutant differs from wild-type SN bytwo amino acid substitutions; Glu was changed to Ser (position 43) andArg was changed to Gly (position 87). SN* folds normally, but has aspecific activity that is 10⁶-fold lower than wild-type SN. Usingtransient expression systems and in trans complementation approaches,fusion protein stability, function and packaging requirements was shown.The present invention shows that Vpr1 and Vpx2 fusion proteins wereexpressed in mammalian cells and were incorporated into HIV particleseven in the presence of wild-type Vpr and/or Vpx proteins. Moreimportantly, however, the present invention shows that virionincorporated Vpr and Vpx fusions remain enzymatically active. Thus,targeting heterologous Vpr and Vpx fusion proteins, includingdeleterious enzymes, to virions represents a new avenue toward anti-HIVdrug discovery.

[0010] In one embodiment of the present invention, there is provided acomposition of matter, comprising: DNA encoding a viral Vpx proteinfused to DNA encoding a virus inhibitory protein.

[0011] In another embodiment of the present invention, there is provideda composition of matter, comprising: DNA encoding a viral Vpr proteinfused to DNA encoding a virus inhibitory protein.

[0012] In yet another embodiment of the present invention, there isprovided a method of delivering a virus inhibitory molecule to a targetin an animal, comprising the step of administering to said animal aneffective amount of the composition of the present invention.

[0013] In still yet another embodiment of the present invention, thereis provided a pharmaceutical composition, comprising a composition ofthe present invention and a pharmaceutically acceptable carrier.

[0014] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted, however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope.

[0016]FIG. 1 shows the construction of vpr1, vpr1SN/SN*, vpx2 andvpx2SN/SN* expression plasmids. FIG. 1A shows the illustration of thepTM-vpr1expression plasmid. The HIV-1_(YU2)vpr coding region wasamplified by PCR and ligated into pTMI at the NcoI and BamHI restrictionsites. FIG. 1B shows the illustration of the pTM-vpx2 expressionplasmid. The HIV-2_(ST)vpx coding region was amplified by PCR andligated into pTMl at the NcoI and Bg1II/SmaI sites. FIG. 1C shows theillustration of the fusion junctions of the pTM-vpr1SN/SN* expressionplasmids. SmaI/XhoI DNA fragments containing SN and SN* were ligatedinto HpaI/XhoI cut pTM-vpr1. Blunt-end ligation at HpaI and Smal siteschanges the vpr translational stop codon (TAA) to Trp and substitutedthe C terminal Ser with a Cys residue. FIG. 1D shows the illustration ofthe fusion junctions of the pTM-vpx2SN/SN* expression plasmids.BanHI/XhoI DNA fragments containing SN and SN* were ligated intoBamHI/Xhol cut pTM-vpx2. In the construction of these plasmids, the VpxC terminal Arg codon was changed to a Val codon and a Ser residue wasintroduced in place of the Vpx translational stop codon (TAA). Fusion ofvpx and SN/SN* at the BamHI sites left a short amino acid sequence ofthe pTM1 polylinker (double underlined) between the two coding regions.

[0017]FIG. 2 shows the expression of Vpr1- and VPX2- SN and SN* fusionproteins in mammalian cells. FIG. 2A shows the pTM1, pTM-vpr1,pTM-vpr1SN and pTM-vpr1SN* were transfected into HeLa cells one hourafter infection with rVT7 (MOI=10). Twenty-four hours later cell lysateswere prepared and examined by immunoblot analysis. Replica blots wereprobed with anti-Vpr1 (left) and anti-SN (right) antibodies. FIG. 2Bshows that replica blots, prepared from rVT7 infected HeLa cellstransfected with pTM1, pTM-vpx2, pTM-vpx2SN and pTM-vpx2SN*, were probedwith anti-Vpx2 (left) and anti-SN (right) antibodies. Bound antibodieswere detected by ECL (Amersham) methods as described by themanufacturer.

[0018]FIG. 3 shows the incorporation of Vpr1- and Vpx2- SN and SN*fusion proteins into virus-like particles (VLP). FIG. 3A transfection ofT7 expressing (rVT7 infected) HeLa cells with pTM-vpr1, pTM-vpr1SN, andpTM-vpr1SN* alone and in combination with pTM-gag1. pTM1 was alsotransfected for control. Culture supernatant were collected twenty-fourhours after transfection, clarified by centrifugation (1000× g, 10 min.)and ultracentrifuged (125,000× g, 2 hrs.) over cushions of 20% sucrose.Pellets (VLPs, middle and bottom panels) and cells (top panel) weresolubilized in loading buffer and examined by immunoblot analysis usinganti-Vpr1 (top and middle) and anti-Gag (bottom) antibodies as probes.FIG. 3B transfection of T7 expressing HeLa cells pTM-vpx2, pTM-vpx2Snand pTM-vpx2SN* alone and in combination with pTM-gag2. Pellets (VLPs,middle and bottom panels) and cells (top panel) were lysed, proteinswere separated by SDS-PAGE and electroblotted blotted to nitrocelluloseas described above. Replica blots were probed with anti-Vpx2 (top andmiddle panels) and anti-Gag (bottom panel) antibodies. Bound antibodieswere detected using ECL methods.

[0019]FIG. 4 shows that virus-specific signals mediate incorporation ofVpr- and Vpx- SN into VLPs. FIG. 4A shows that HIV-1 Gag mediatespackaging of Vpr1SN. rVT7 infected (T7 expressing) HeLa cells weretransfected with pTM-vpr1SN alone and in combination with pTM-gag2 andpTM-gag1. Pellets (VLPs, middle and bottom panels) and cells (top panel)were prepared 24 hours after transfection and examined by immunoblotanalysis using anti-Vpr1 (top and middle) and anti-Gag (bottom)antibodies for probes. (B) HIV-2 Gag mediates packaging of Vpx2SN. T7expressing HeLa cells were transfected with pTM-vpx2SN alone and incombination with pTM-gag1 and pTM-gag2. Pellets (VLPs, middle and bottompanels) and cells (top panel) were prepared 24 hours after transfectionand examined by immunoblot analysis using anti-Vpx2 (top and middle) andanti-Gag (bottom) antibodies for probes.

[0020]FIG. 5 shows a competition analysis of Vpr1SN and Vpx2SN forincorporation into VLPs. FIG. 5A shows transfection of T7 expressingHeLa cells with different amounts of pTM-vpr1 (2.5, 5 and 10 ug) andpTM-vpr1SN (2.5, 5 and 10 ug), either individually or together incombination with pTM-gag1 (10 ug). FIG. 5B shows that HeLa cells weretransfected with different amounts of pTM-vpx2 (2.5, 5 and 10 ug) andpTM-vpx2SN (2.5, 5 and 10 ug), either individually or together withpTM-gag2 (10 ug). Twenty hours after transfection, particles wereconcentrated by ultracentrifugation through sucrose cushions andanalyzed by immunoblotting using anti-Vpr1 (A) or anti-Vpx2 (B)antibodies.

[0021]FIG. 6 shows the nuclease activity of VLP-associated Vpr1SN andVpx2SN proteins. Virus-like particles were concentrated from culturesupernatants of T7 expressing HeLa cells cotransfected withpTM-gag1/pTM-vpr1SN, pTM-gag1/pTM-vpr1SN*, pTM-gag2/pTM-vpx2SN andpTM-gag2/pTM-vpx2SN* by ultracentrifugation (125,000× g, 2 hrs.) through20% cushions of sucrose. Pellets containing Vpr1-SN and SN* (B) andVpx2- SN and SN* (C) were resuspended in PBS. Tenfold dilutions weremade in nuclease reaction cocktail buffer (100 mM Tris-HCI pH 8.8, 10 mMCaCl₂, 0.1% NP40) and boiled for 1 minute. 5 ul of each dilution wasadded to 14 ul of reaction cocktail buffer containing 500 ng of lambdaphage DNA (HindIII fragments) and incubated at 37° C. for 2 hours.Reaction products were electrophoresed on 0.8% agarose gels and DNA wasvisualized by ethidium bromide staining. Standards (A) were prepared bydilution of purified staphylococcal nuclease (provided by A. Mildvan)into cocktail buffer and assayed.

[0022]FIG. 7 shows the incorporation of Vpx2SN into HIV-2 by transcomplementation. FIG. 7A shows the construction of the pLR2P-vpx2SN/SN*expression plasmids. To facilitate efficient expression of HIV genes,the HIV-2 LTR and RRE were engineered into the polylinker of pTZ19U,generating pLR2P. The organization of these elements within the pTZ19Upolylinker is illustrated. Ncol/Xhol vpx2SN and vpx2SN* (vpxSN/SN*)containing DNA fragments were ligated into pLR2P, generatingpLR2P-vpx2SN and pLR2P-vpx2SN* (pLR2P-vpxSN/SN*). FIG. 7B shows theassociation of Vpx2SN with HIV-2 virions. Monolayer cultures of HLtatcells were transfected with HIV-2ST proviral DNA (PSXB1) andcotransfected with pSXB1/pTM-vpx2SN and pSXB1/pTM-vpx2SN. Extracellularvirus was concentrated from culture supernatants forty-eight hours aftertransfection by ultracentrifugation (125,000× g, 2 hrs.) throughcushions of 20% sucrose. Duplicate Western blots of viral pellets wereprepared and probed independently with anti-Vpx2 (left) anti-SN(middle)-and anti-Gag (right) antibodies. FIG. 7C shows a sucrosegradient analysis. Pellets of supernatant-virus prepared frompSXB1/pTM-vpx2SN cotransfected HLtat cells were resuspended in PBS,layered over a 20-60% linear gradient of sucrose and centrifuged for 18hours at 125,000× g. Fractions (0.5 ml) were collected from the bottomof the tube, diluted 1:3 in PBS, reprecipitated and solubilized inelectrophoresis buffer for immunoblot analysis. Replica blots wereprobed with anti-SN (top) and anti-Gag (bottom) antibodies. Fraction 1represents the first collection from the bottom of the gradient andfraction 19 represents the last collection. Only alternate fractions areshown, except at the peak of protein detection. FIG. 7D shows theincorporation of Vpx2SN into HIV-2_(7312A) Vpr and Vpx competent virus.Virus concentrated from supernatants of HLtat cells transfected withHIV-2_(7312A) proviral DNA (pJK) or cotransfected with pJK/pLR2P-vpx2SNor pJK/pLR2P-vpx2SN* was prepared for immunoblot analysis as describedabove. Included for control were virions derived by pSXB1/pLR2P-vpx2SN*cotransfection. Duplicate blots were probed with anti-Vpx (left) andanti-Gag (right) antibodies.

[0023]FIG. 8 shows the incorporation of Vpr1 SN into HIV-1 virions bytrans complementation. Culture supernatant virus from HLtat cellstransfected with pNL4-3 (HIV-1) and pNL4-3R (HIV-1 vpr mutant) orcotransfected with pNL4-3/pLR2P-vpr1SN and pNL4-3R/pLR2P-vpr1SN wasprepared for immunoblot analysis as described above. Blots were probedwith anti-SN (FIG. 8A), anti-Vpr1(FIG. 8B) and anti-Gag (FIG. 8C)antibodies.

[0024]FIG. 9 shows the inhibition of Vpr1/Vpx2-SN processing by an HIVprotease inhibitor. HIV-1 (pSG3) and HIV-2 (pSXB1) proviral DNAs werecotransfected separately into replica cultures of HLtat cells withpLR2P-vpr1SN and pLR2P-vpx2SN, respectively. One culture of eachtransfection contained medium supplemented with 1 uM of the HIV proteaseinhibitor L-699-502. Virions were concentrated from culture supernatantsby ultracentrifugation through cushions of 20% sucrose and examined byimmunoblot analysis using anti-Gag (FIG. 9A) and anti-SN (FIG. 9B)antibodies.

[0025]FIG. 10 shows the incorporation of enzymatically active Vpr1- andVpx2-CAT fusion proteins into HIV virions. FIG. 10A shows anillustration of the fusion junctions of the pLR2P-vpr1CAT andpLR2P-vpx2CAT expression plasmids. PCR amplified BamHI/XhoI DNAfragments containing CAT were ligated into Bg1II/XhoI cut pLR2P-vpr1SNand pLR2P-vpx2SAN, replacing SN (see FIG. 1). This constructionintroduced two additional amino acid residues (Asp and Leu, aboveblackened bar) between the vpr1/vpx2CAT coding regions. FIG. 10B showsthe incorporation of Vpr1CAT into HIV-1 virions. Virus produced fromHLtat cells transfected with pNL4-3 (HIV-1) and pNL4-3R (HIV-1R), orcotransfected with pNL4-3/pLR2P-vpr1CAT and pNL4-3R/pLR2P-vpr1CAT wasprepared as described above and examined by immunoblot analysis. Replicablots were probed with anti-Vpr1 (left) and anti-Gag (right) antibodies.FIG. 10C shows the incorporation of Vpx2CAT into HIV-2 virions. Virusproduced from HLtat cells transfected with pSXB1 (HIV-2) orcotransfected with pSXB1/pLR2P-vpx2CAT was prepared as described aboveand examined by immunoblot analysis. Replica blots were probed withanti-Vpx2 (left) and anti-Gag (right) antibodies. FIG. 10D shows thatvirion incorporated Vpr1- and Vpx2- CAT fusion proteins possessenzymatic activity. Viruses pelleted from HLtat cells transfected withpSXB1 (HIV-2) or cotransfected with pSXB1/pLR2P-vpx2CAT andpNL4-3/pLR2P-vpr1CAT were lysed and analyzed for CAT activity. HIV-2 wasincluded as a negative control.

[0026]FIG. 11 shows virion association of enzymatically active CAT andSN fusion proteins. FIG. 11A shows that HIV-2 virions collected from theculture supernatant of HLtat cells cotransfected with pSXB1 andpLR2P-vpx2 were sedimented in linear gradients of 20-60% sucrose. 0.7 mlfractions were collected and analyzed by immunoblot analysis using Gagmonoclonal antibodies as a probe. FIG. 11B shows CAT enzyme activity wasdetermined in each fraction by standard methods. The positions ofnonacetylated [¹⁴C]chloramphenicol (Cm) and acetylated chloramphenicol(Ac-Cm) are indicated. FIG. 11C shows HIV-1 virions derived from HLtatcells cotransfected with pSG3 and pLR2P-vpr1SN and cultured in thepresence of L-689,502 were sedimented in linear gradients of 20-60%sucrose. Fractions were collected and analyzed for virus content byimmunoblot analysis using Gag monoclonal antibodies. FIG. 11D shows thatSN activity was determined in each fraction as described in FIG. 6.

DETAILED DESCRIPTION OF THE INVENTION

[0027] As used herein, the term “fusion protein” refers to either theentire native protein amino acid sequence of Vpx (ofany HIV-2 and SIV)and Vpr (of any HIV-1 and SIV) or any subfraction of their sequencesthat have been joined through recombinant DNA technology and are capableof association with either native HIV/SIV virions or virus likeparticles.

[0028] As used herein, the term “virion” refers to HIV-1, HIV-2 and SIVvirus particles.

[0029] As used herein, the term “virus-like particle” refers to anycomposition of HIV-1, HIV-2 and SIV proteins other than which existsnaturally in naturally infected individuals or monkey species that arecapable of assembly and release from either natural or immortalizedcells that express these proteins.

[0030] As used herein, the term “transfect” refers to the introductionof nucleic acids (either DNA or RNA) into eukaryotic or prokaryoticcells or organisms.

[0031] As used herein, the term “virus-inhibitory protein” refers to anysequence of amino acids that have been fused with Vpx or Vpr sequencesthat may alter in any way the ability of HIV-1, HIV-2 or SIV viruses tomultiply and spread in either individual cells (prokaryotic andeukaryotic) or in higher organisms. Such inhibitory molecules mayinclude: HIV/SIV proteins or sequences, including those that may possessenzymatic activity (examples may include the HIV/SIV protease,integrase, reverse transcriptase, Vif, Nef and Gag proteins) HIV/SIVproteins or proteins/peptide sequences that have been modified bygenetic engineering technologies in order to alter in any way theirnormal function or enzymatic activity and/or specificity (examples mayinclude mutations of the HIV/SIV protease, integrase, reversetranscriptase, Vif, Nef and Gag proteins), or any other non viralprotein that, when expressed as a fusion protein with Vpx or Vpr, altervirus multiplication and spread in vitro or in vivo.

[0032] In the present invention, the HIV Vpr and Vpx proteins werepackaged into virions through virus type-specific interactions with theGag polyprotein precursor. HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) areutilized to target foreign proteins to the HIV particle as their openreading frames were fused in-frame with genes encoding the bacterialstaphylococcal nuclease (SN), an enzymatically inactive mutant of SN(SN*), and the chloramphenicol acetyl transferase (CAT). Transientexpression in a T7-based vaccinia virus system demonstrated thesynthesis of appropriately sized Vpr1 SN/SN* and Vpx2SN/SN* fusionproteins which, when co-expressed with their cognate p55^(Gag) protein,were efficiently incorporated into virus-like particles (VLPs).Packaging of the fusion proteins was dependent on virus type-specificdeterminants, as previously seen with wild-type Vpr and Vpx proteins.Particle associated Vpr1SN and Vpx2SN fusion proteins were enzymaticallyactive as determined by in vitro digestion of lambda phage DNA. Todemonstrate that functional Vpr1 and Vpx2 fusion proteins were targetedto HIV particles, the gene-fusions were cloned into an HIV-2 LTR/RREregulated expression vector and co-transfected with wild-type HIV-1 andHIV-2 proviruses. Western blot analysis of sucrose gradient purifiedvirions revealed that both Vpr1 and Vpx2 fusion proteins wereefficiently packaged regardless of whether SN, SN* or CAT were used as Cterminal fusion partners. Moreover, the fusion proteins remainedenzymatically active and were packaged in the presence of wild-type Vprand Vpx proteins. Interestingly, virions also contained smaller sizedproteins that reacted with antibodies specific for the accessoryproteins as well as SN and CAT fusion partners. Since similar proteinswere absent from Gag-derived VLPs as well as in virions propagated inthe presence of an HIV protease inhibitor, they must represent cleavageproducts produced by the viral protease. Taken together, these resultsdemonstrate that Vpr and Vpx can be used to target functional proteins,including potentially deleterious enzymes, to the HIV/SIV particle.These properties are useful for the development of novel antiviralstrategies.

[0033] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1 Cells and Viruses

[0034] HeLa, HeLa-tat (HLtat) and CV-1 cells were maintained inDulbecco's Modified Eagle's Medium supplemented with 10% fetal bovineserum (FBS). HLtat cells constitutively express the first exon of HIV-1tat and were provided by Drs. B. Felber and G. Pavlakis. A recombinantvaccinia virus (rVT7) containing the bacteriophage T7 RNA polymerasegene was used to facilitate expression of viral genes placed under thecontrol of a T7 promoter. Stocks of rVT7 were prepared and titrated inCV-1 cells as described previously by Wu, et al., J. Virol. 66:7104-7112(1992). HIV-1_(YU2), HIV-1 pNL 4-3-R and pNL 4-3, HIV-1_(HXB2D),HIV-2_(ST), and HIV-2_(7312A) proviral clones were used for theconstruction of recombinant expression plasmids and the generation oftransfection derived viruses.

EXAMPLE 2 Antibodies

[0035] To generate HIV-1 Vpr specific antibodies, the HIV-1_(YU-2)vpropen reading frame was amplified by polymerase chain reaction (PCR)using primers (sense: 5′GCCACCTTTGTCGACTGTTAAAAAACT-3′ and anti-sense:5′-GTCCTAGGCAAGCTTCCTGGATGC-3′) containing Sa1I and Hind1II sites andligated into the prokaryotic expression vector, pGEX, generatingpGEX-vpr1. This construct allowed expression of Vpr1 as a C terminalfusion protein and glutathione S-transferase (gst), thus allowingprotein purification using affinity chromatography. E. coli (DH5a) weretransformed with pGEX-vpr1 and protein expression was induced withisopropyl β-D thiogalactopyranoside (IPTG). Expression of the gst-Vpr1fusion protein was confirmed by SDS-PAGE. Soluble gst-Vpr1 protein waspurified and Vpr1 was released by thrombin cleavage using previouslydescribed procedures of Smith, et al., Gene 67:31-40 (1988). New ZealandWhite rabbits were immunized with 0.4 mg of purified Vpr1 proteinemulsified 1:1 in Freunds complete adjuvant, boosted three times at twoweek intervals with 0.25 mg of Vpr1 mixed 1:1 in Freunds' incompleteadjuvant and bled eight and ten weeks after the first immunization tocollect antisera. Additional antibodies used included monoclonalantibodies to HIV-1 Gag (ACT1, and HIV-2 Gag (6D2.6), polyclonal rabbitantibodies raised against the HIV-2 Vpx protein and anti-SN antiserumraised against purified bacterially expressed SN protein.

EXAMPLE 3 Construction of T7-Based Expression Plasmids

[0036] A DNA fragment encompassing ^(HIV-1)HXB2D^(gag) (nucleotides335-1837) was amplified by PCR using primers (sense:5′-AAGGAGAGCCATGGGTGCGAGAGCG-3′ and anti-sense:5′GGGGATCCCTTTATTGTGACGAGGGG-3′) containing NcoI and BamHI restrictionsites (underlined). The PCR product was digested with NcoI and BamHI,purified and ligated into the polylinker of the pTM1 vector, generatingpTM-gag1. Similarly, a DNA fragment containing the gag coding region ofHIV-2_(ST) (nucleotides 547-2113) was amplified by PCR using sense andanti-sense primers 5′-ATTGTGGGCCATGGGCGCGAGAAAC-3′ and5′GGGGGGCCCCTACTGGTCTTTTCC-3′, respectively. The reaction product wascut with NcoI and SmaI (underlined), purified and ligated into thepolylinker of pTM1, generating pTM-gag2.

[0037] For expression of Vpr1 under the control of the T7 promoter, aDNA fragment containing the HIV-1_(YU2)vpr coding region (nucleotides5107-5400) was amplified by PCR using primers (sense:5′-GAAGATCTACCATGGAAGCCCCAGAAGA-3′ and anti-sense:5′-CGCGGATCCGTTAACATCTACTGGCTCCATTTCTTGCTC-3′) containing NcoI andHpaI/BamHI sites, respectively (underlined). The reaction product wascut with NcoI and BamHI and ligated into pTM1, generating a pTM-vpr1(FIG. 12A). In order to fuse SN and SN* in-frame with vpr1, their codingregions were excised from pGN1561.1 and pGN1709.3, respectively andthrough a series of subcloning steps, ligated into the SmaI/XhoI sitesof pTM-vpr1, generating pTM-vpr1SN and pTM-vpr1SN*. This approachchanged the translational stop codon of Vpr1 to a Trp codon and the Cterminal Ser residue to a Cys. The resulting junctions between vpr1 andSN/SN* are depicted in FIG. 1C.

[0038] For expression of Vpx2 under T7 control, a DNA fragmentcontaining the HIV-2_(ST) vpx coding sequence (nucleotides 5343-5691)was amplified by PCR using primers (sense:5′GTGCAACACCATGGCAGGCCCCAGA-3′ and anti-sense:5′-TGCACTGCAGGAAGATCTTAGACCTGGAGGGGGAGGAGG-3′) containing NcoI and Bg1IIsites, respectively (underlined). After cleave with Bg1II and Klenowfill-in, the PCR product was cleaved with NcoI, purified and ligatedinto the NcoI and SmaI sites of pTM1, generating pTM-vpx2 (FIG. 1B). Toconstruct in-frame fusions with vpx2, BamHI/XhoI, SN- and SN*-containingDNA fragments were excised from pTM-vpr1SN and pTM-vpr1SN* and ligatedinto pTM-vpx2, generating pTM-vpx2SN and pTM-vpx2SN*, respectively. Thisapproach introduced one amino acid substitution at the C terminus of Vpx(Val to Arg), changed the translational stop codon of vpx to Ser andleft five amino acids residues of the pTM1 plasmid polylinker. Theresulting junctions between vpx2 and SN/SN* are depicted in FIG. 1D.

EXAMPLE 4 Construction of HIV LTR-Based Expression Plasmids

[0039] For efficient expression of Vpr and Vpx fusion proteins in thepresence of HIV, a eukaryotic expression vector (termed pLR2P) wasconstructed which contains both an HIV-2 LTR (HIV-2_(ST), coordinates-544 to 466) and an HIV-2 RRE (HIV-2_(ROD), coordinates 7320 to 7972)element (FIG. 7A). These HIV-2 LTR and RRE elements were chosen becausethey respond to both HIV-1 and HIV-2 Tat and Rev proteins. The vpr1,vpr1SN, vpx2 and vpx2SN coding regions were excised from theirrespective pTM expression plasmids (see FIG. 1) with NcoI and XhoIrestriction enzymes and ligated into pLR2P, generating pLR2P-vpr1,pLR2P-vpr1SN, pLR2P-vpx2 and pLR2P-vpx2SN, respectively (FIG. 7A). Forconstruction and expression of vpr- and vpx- CAT gene fusions, the SNcontaining regions (BamHI/XhoI fragments) of pLR2P-vpr1SN andpLR2P-vpx2SN were removed and substituted with a PCR amplifiedBg1II/XhoI DNA fragment containing CAT, generating pLR2P-vpr1CAT andpLR2P-vpx2CAT, respectively (FIG. 9A).

EXAMPLE 5 Transfections

[0040] Transfections of proviral clones were performed in HLtat cellsusing calcium phosphate DNA precipitation methods as described by themanufacturer (Strategene). T7-based (pTMI) expression constructs weretransfected using Lipofectin (BioRad) into rVT7 infected HeLa cells asdescribed previously by Wu, et al., J. Virol. 68:6161-6169 (1994). Thesemethods were those recommended by the manufacturer of the Lipofectinreagent.

EXAMPLE 6 Western Immunoblot Analysis

[0041] Virions and virus-like particles (VLPs) were concentrated fromthe supernatants of transfected or infected cells by ultracentrifugationthrough 20% cushions of sucrose (125,000× g, 2 hrs., 4° C.). Pellets andinfected/transfected cells were solubilized in loading buffer [62.5 mMTris-HCl (pH 6.8) 0.2% sodium lauryl sulfate (SDS), 5%2-mercaptoethanol, 10% glycerol], boiled and separated on 12.5%polyacrylamide gels containing SDS. Following electrophoresis, proteinswere transferred to nitrocellulose (0.2 μm; Schleicher & Schuell) byelectroblotting, incubated for one hour at room temperature in blockingbuffer (5% nonfat dry milk in phosphate buffered saline [PBS]) and thenfor two hours with the appropriate antibodies diluted in blockingbuffer. Protein bound antibodies were detected with HRP-conjugatedspecific secondary antibodies using ECL methods according to themanufacturer's instructions (Amersham).

EXAMPLE 7 SN Nuclease Activity Assay

[0042] Cells and viral pellets were resuspended in nuclease lysis buffer(40 mM Tris-HCl, pH 6.8, 100 mM NaCl, 0.1% SDS, 1% Triton X-00) andclarified by low speed centrifugation (1000× g, 10 min.). Tenfolddilutions were made in nuclease reaction cocktail buffer (100 mMTris-HCl, pH 8.8, 10 mM CaCl₂, 0.1% NP40) and boiled for 1 minute. 5 μlof each dilution was added to 14 μ1 of reaction cocktail buffercontaining 500 ng of lambda phage DNA (Hind1II fragments) and incubatedat 37° C. for 2 hours. Reaction products were electrophoresed on 0.8%agarose gels and DNA was visualized by ethidium bromide staining.

EXAMPLE 8 Expression of Vpr1- and Vpx2- SN and SN* Fusion Proteins inMammalian Cells

[0043] Expression of Vpr1- and Vpx2- SN/SN* fusion proteins in mammaliancells was assessed using the recombinant vaccinia virus-T7 system(rVT7). HeLa cells were grown to 75-80% confluency and transfected withthe recombinant plasmids pTM-vpr, pTM-vpx, pTM-vpr1SN/SN*, andpTM-vpx2SN/SN* (FIG. 1). Twenty-four hours after transfection, cellswere washed twice with PBS and lysed. Soluble proteins were separated bySDS-PAGE and subjected to immunoblot blot analysis. The results areshown in FIG. 2. Transfection of pTM-vpr1SN and pTM-vpr1SN* resulted inthe expression of a 34 kDa fusion protein that was detectable using bothanti-Vpr and anti-SN antibodies (A). Similarly, transfection ofpTM-vpx2SN and pTM-vpx2SN* resulted in the expression of a 35 kDa fusionprotein which was detected using anti-Vpx and anti-SN antibodies (B).Both fusion proteins were found to migrate slightly slower thanexpected, based on the combined molecular weights of Vpr1 (14.5 kDa) andSN (16 kDa) and Vpx2 (15 kDa) and SN, respectively. Transfection ofpTM-vpr1 and pTM-vpx2 alone yielded appropriately sized wild-type Vprand Vpx proteins. Anti-Vpr, anti-Vpx and anti-SN antibodies were notreactive with lysates of pTM1 transfected cells included as controls.Thus, both SN and SN* fusion proteins can be expressed in mammaliancells.

EXAMPLE 9 Incorporation of Vpr1- and Vpr2- SN/SN* Fusion Proteins IntoVirus-Like Particles.

[0044] In vaccinia and baculovirus systems, the expression of HIV Gag issufficient for assembly and extracellular release of VLPs. Vpr1 and Vpx2can be efficiently incorporated into Gag particles without theexpression of other viral gene products. To demonstrate that the Vpr1and Vpx2 fusion proteins could be packaged into VLPs, recombinantplasmids were coexpressed with HIV-1 and HIV-2 Gag proteins in the rVT7system. pTM-vpr1, pTM-vpr1SN and pTM-vpr1SN* were transfected into HeLacells alone and in combination with the HIV-1 Gag expression plasmid,pTM-gag1. Twenty-four hours after transfection, cell and VLP extractswere prepared and analyzed by immunoblot analysis (FIG. 3A). Anti-Vprantibody detected Vpr1, Vpr1SN and Vpr1SN* in cell lysates (top panel)and in pelleted VLPs derived by coexpression with pTM-gag1 (middlepanel). In the absence of HIV-1Gag expression, Vpr1 and Vpr1SN were notdetected in pellets of culture supernatants (middle panel). As expectedVLPs also contained p55 Gag (bottom panel). Thus, Vpr1SN/SN* fusionproteins were successfully packaged into VLPs.

[0045] To demonstrate that Vpx2SN was similarly capable of packaginginto HIV-2 VLPs, pTM-vpx2, pTM-vpx2SN and pTM-vpx2SN* were transfectedinto HeLa cells alone and in combination with the HIV-2 Gag expressionplasmid, pTM-gag2. Western blots were prepared with lysates of cells andVLPs concentrated from culture supernatants by ultracentrifugation (FIG.3B). Anti-Vpx antibody detected Vpx2, Vpx2SN and Vpx2SN* in cell lysates(top panel) and in VLPs derived by coexpression with pTM-gag2 (middlepanel). Anti-Gag antibody detected p55 Gag in VLP pellets (bottompanel). Comparison of the relative protein signal intensities suggestedthat the Vpr1- and Vpx2- SN and SN* fusion proteins were packaged intoVLPs in amounts similar to wild-type Vpr1 and Vpx2 proteins. Sucrosegradient analysis of VLPs containing Vpr1SN and Vpx2SN demonstratedco-sedimentation of these fusion proteins with VLPs (data not shown).

[0046] The Gag C terminal region is required for incorporation of Vpr1and Vpx2 into virions. However, packaging was found to be virustype-specific, that is, when expressed in trans, Vpx2 was onlyefficiently incorporated into HIV-2 virions and HIV-2 VLPs. Similarly,HIV-1 Vpr required interaction with the HIV-1 Gag precursor forincorporation into HIV-1 VLPs. To show that the association of Vpr1SNand Vpx2SN with VLPs was not mediated by the SN moiety, but was due tothe Vpr and Vpx specific packaging signals, pTM-vpr1SN and pTM-vpx2SNwere cotransfected individually with either pTM-gag1 or pTM-gag2. Forcontrol, pTM-vpr1 and pTM-vpx2 were also transfected alone. Twenty-fourhours later, lysates of cells and pelleted VLPs were examined byimmunoblotting (FIG. 4). While Vpr1SN was expressed in all cells (FIG.4A, top panel), it was only associated with VLPs derived from cellstransfected with pTM-gag1. Similarly, Vpx2SN was detected in allpTM-vpx2 transfected cells (FIG. 4B, top panel), but was only associatedwith VLPs derived by cotransfection with pTM-gag2 (FIG. 4B, middlepanel). HIV-1 and HIV-2 Gag monoclonal antibodies confirmed the presenceof Gag precursor protein in each VLP pellet (FIG. 4B, bottom panels).Thus, incorporation of Vpr1SN and Vpx2SN into VLPs requires interactionof the cognate Gag precursor protein, just like native Vpr1 and Vpx2.

[0047] While Vpr1SN and Vpx2SN fusion proteins clearly associated withVLPs (FIG. 3), the question remained whether they would continue to doso in the presence of the native accessory proteins. The efficiency ofVpr1SN and Vpx2SN packaging was compared by competition analysis (FIG.5). pTM-vpr1SN and pTM-vpx2SN were cotransfected with pTM-gag1/pTM-vpr1and pTMgag2/pTM-vpx2, respectively, using ratios that ranged from 1:4 to4:1 (FIG. 5A and FIG. 5B, left panels). For comparison, pTM-vpr1SN andpTM-vpr1 were transfected individually with pTM-gag1 (FIG. 5A, middleand right panels respectively) and pTM-vpx2SN and pTM-vpx2 weretransfected with pTM-gag2 (FIG. 5B, middle and right panelsrespectively). VLPs were pelleted through sucrose cushions, lysed,separated by PAGE, blotted onto nitrocellulose and probed with anti-SNantibody. The results revealed the presence of both Vpr1 and Vpr1SN inVLPs when cotransfected into the same cells (FIg. 5A, left panel).Similarly, coexpressed Vpx2 and Vpx2SN were also copackaged (FIG. 5B,left panel). Comparison of the relative amounts of VLP-associated Vpr1SNand Vpx2SN when expressed in the presence and absence of the nativeprotein, indicated that there were no significant packaging differences.Thus, Vpr1/Vpx2 fusion proteins can efficiently compete with wild-typeproteins for virion incorporation.

EXAMPLE 10 Vpr1SN and Vpx2SN Fusion Proteins Possess Nuclease Activity

[0048] To demonstrate that virion associated SN fusion proteins wereenzymatically active, VLPs concentrated by ultracentrifugation fromculture supernatants of HeLa cells transfected with pTM-gag1/pTM-vpr1SNand pTM-gag2/pTM-vpx2SN were analyzed for nuclease activity using an invitro DNA digestion assay. Prior to this analysis, immunoblottingconfirmed the association of Vpr1SN and Vpx2SN with VLPs (data notshown). FIG. 6 shows lambda phage DNA fragments in 0.8% agarose gelsafter incubation with dilutions of VLPs lysates that contained Vpr1- orVpx2-SN fusion proteins. VLPs containing Vpr1SN* and Vpx2SN* wereincluded as negative controls and dilutions of purified SN served asreference standards (FIG. 6A). Both virion associated Vpr1SN (FIG. 6B)and Vpx2SN (FIG. 6C) fusion proteins exhibited nuclease activity asdemonstrated by degradation of lambda phage DNA. Cell-associated Vpr1SNand Vpx2SN fusion proteins also possessed nuclease activity whenanalyzed in this system (data not shown). To control for SN specificity,this analysis was also conducted in buffers devoid of Ca⁺⁺ and underthese conditions no SN activity was detected (data not shown). Thus, SNremains enzymatically active when expressed as a fusion protein andpackaged into VLPs.

EXAMPLE 11 Incorporation of Vpx2SN Fusion Protein Into HIV-2 Virions

[0049] Vpx is incorporated into HIV-2 virions when expressed in trans.To show that Vpx2 fusion proteins were similarly capable of packaginginto wild-type HIV-2 virions, an expression plasmid (pLR2P) wasconstructed placing the vpx2SN and vpx2SN* coding regions under controlof HIV-2 LTR and RRE elements. The HIV-2 RRE was positioned downstreamof the fusion genes to ensure mRNA stability and efficient translation(FIG. 7A). To show that the fusion proteins could package when expressedin trans, HIV-2_(ST) proviral DNA (pSXBI) was transfected alone and incombination with pLR2P-vpx2SN and pLR2P-vpx2SN*. Forty-eight hourslater, extracellular virus as pelleted from culture supernatants byultracentrifugation through cushions of 20% sucrose and examined byimmunoblot analysis (FIG. 7B). Duplicate blots were probed usinganti-Vpx (left), anti-SN (middle) and anti-Gag (right) antibodies.Anti-Vpx antibody detected the 15 kDa Vpx2 protein in all viral pellets.In virions derived by cotransfection of HIV-2_(ST) with pLR2P-vpx2SN andpLR2P-vpx2SN*, additional proteins of approximately 35 and 32 kDa wereclearly visible. The same two proteins were also apparent on a duplicateblot probed with anti-SN antibodies, indicating that they representedVpx2SN fusion proteins (FIG. 7B, middle panel). The predicted molecularweight of full-length Vpx2SN fusion protein is 33 kDa As native Vpx andSN run slightly slower than predicted, it is likely that the 35 kDaspecies represents the full-length Vpx2SN fusion protein. Anti-SNantibodies detected additional proteins of approximately 21 and 17 kDa(these proteins were more apparent after longer exposure). Since onlythe 35 kDa protein was detected in Gag derived VLPs, which lack Polproteins (FIG. 2), the smaller proteins represented cleavage products ofVpx2SN and Vpx2SN* generated by the viral protease. Anti-Gag antibodiesconfirmed the analysis of approximately equivalent amounts of virionsfrom each transfection.

[0050] To show packaging of Vpx2SN into HIV-2 virions, sucrose gradientanalysis was performed. Extracellular virus collected from culturesupernatants of HLtat cells forty-eight hours after cotransfection withpLR2P-vpx2SN and HIV-2_(ST) was pelleted through cushions of 20%sucrose. Pellets were resuspended in PBS and then centrifuged for 18hours over linear gradients of 20-60% sucrose. Fractions were collectedand analyzed by immunoblotting (FIG. 7C). Duplicate blots were probedseparately with anti-SN (top) and anti-Gag (bottom) antibodies. Peakconcentrations of both Vpx2SN and Gag were detected in fractions 8-11,demonstrating direct association and packaging of Vpx2SN into HIV-2virions. These same sucrose fractions (8-11) were found to havedensities between 1.16 and 1.17 g/ml, as determined by refractometricanalysis (data not shown). Again, both the 35 kDa and 32 kDa forms ofVpx2SN were detected, providing further evidence for protease cleavagefollowing packaging into virus particles.

[0051] Since HIV-2ST is defective in vpr, this may have affected thepackaging of the Vpx2SN fusion protein. A second strain of HIV-2, termedHIV-_(27312A), was analyzed which was cloned from short-term PBMCculture and contains open reading frames for all genes, including intactvpr and vpx genes (unpublished). A plasmid clone of HIV-2_(7312A)proviral DNA (pJK) was transfected alone and in combination withpLR2P-vpx2SN into HLtat cells. For comparison, HIV-2ST was alsoco-transfected with pLR2P-vpx2SN. Progeny virus was concentrated byultracentrifugation through sucrose cushions and examined by immunoblotanalysis (FIG. 7D). Duplicate blots were probed with anti-Vpx (left) andanti-Gag (right) antibodies. The results revealed comparable levels ofVpx2SN incorporation into vpr competent virus (HIV-2_(7312A)) comparedwith vpr-defective virus (HIV-2sT). Moreover, the 35 kDa and 32 kDaproteins were again detected in HIV-2_(7312A) virions. Thus, efficientincorporation of the Vpx2SN protein into replication-competent wild-typeHIV-2 was demonstrated, even in the presence of native Vpr and Vpxproteins.

EXAMPLE 12 Incorporation of Vpr1SN Into HIV-1 Virions

[0052] Using the same LTR/RRE-based expression plasmid, it was alsoshown that Vpr1SN could package into HIV-1 virions by co-expression withHIV-1 provirus (as discussed above, the HIV-2 LTR can be transactivatedby HIV- 1 Tat and the HIV-2 RRE is sensitive to the HIV-1 Rev protein).Virions released into the culture medium 48 hours after transfection ofHLtat cells with pNL4-3 (HIV-1) and pNL4-3-R (HIV-1-R) alone and incombination with pLR2P-vpr1SN were concentrated by ultracentrifugationand examined by immunoblot analysis (FIG. 8). As observed incotransfection experiments with HIV-2, anti-SN antibodies identified twomajor Vpr1SN fusion proteins of approximately 34 to 31 kDa. Theseproteins were not detected in virions produced by transfection of pNL4-3and pNL4-e-R alone. From expression in the rVT7 system, the full-lengthVpr1SN fusion protein was expected to migrate at 34 kDa. Therefore, the31 kDa protein likely represents a cleavage product. Anti-SN antibodiesalso detected a protein migrating at 17 kDa. Anti-Vpr antibody detectedthe 34 and 31 kDa proteins in virions derived from cotransfected cells.It is noteworthy that both the anti-Vpr and anti-SN antibodies detectedthe 31 kDa protein most strongly, and that anti-Vpr antibody did notdetect the 17 kDa protein recognized by anti-SN antibody. These resultsalso show that even in virions in which native Vpr protein was packaged,Vpr1SN was also incorporated in abundance. Gag monoclonal antibodydetected similar amounts of Gag protein in all viral pellets anddemonstrated processing of the p55^(Gag) precursor (FIG. 8C).

[0053] To demonstrate more directly that cleavage of the Vpr1and Vpx2-SNfusion proteins was mediated by the HIV protease, virus was concentratedfrom pNL4-3-R/pLR2P-vpr1SN and pSXB1/pLR2P-vpx2SN transfected cells thatwere culture in the presence of 1 μM of the HIV protease inhibitorL-689,502 (provided by Dr. E. Emini, Merck & Co. Inc.). As expected,immunoblot analysis of virions demonstrated substantially lessprocessing of p55^(Gag) (FIG. 9A). Similarly, virions produced in thepresence of L-689,502 also contained greater amounts of the uncleavedspecies of Vpr1SN and Vpx2SN fusion proteins (FIG. 9B). Taken together,these results demonstrate that Vpr1- and Vpx2-SN fusion proteins aresubject to protease cleavage during or subsequent to virus assembly.

EXAMPLE 13 Vpr1-CAT and Vpr2-CAT Fusion Protein Incorporation Into HIVVirions.

[0054] To show that Vpx2 and Vpr1 could target additional proteins tothe HIV particle, the entire 740 bp CAT gene was substituted for SN inthe pLR2P-vpx2SN and pLR2P-vpr1SN vectors, generating pLR2P-vpr1 CAT andpLR2P-vpx2CAT (FIG. 10A). pNL4-3/pLR2P-vpr1CAT, pnl4-3-R/pLR2P-vpr1CATand pSXB1/pLR2P-vpx2CAT were co-transfected into HLtat cells. Ascontrols, pNL4-3, pNL4-3-R and pSXB1 were transfected alone. Progenyvirions, concentrated from culture supernatants, were analyzed byimmunoblotting (FIGS. 10B and 10C). Using anti-Vpr antibodies, 40 kDafusion proteins were detected in viral pellets derived byco-transfection of pRL2P-vpr1CAT with both pNL4-3 and pNL4-3-R (FIG.10B). This size is consistent with the predicted molecular weight of thefull-length Vpr1CAT fusion protein. In addition, anti-Vpr antibodiesalso detected a 17 kDa protein which did not correspond to the molecularweight of native Vpr1 protein (14.5 kDa in virions derived from cellstransfected with pNL4-3). The same protein was recognized weakly withanti-CAT antibodies, suggesting a fusion protein cleavage productcontaining most Vpr sequence. Very similar results were obtained withvirions derived from HLtat cells co-transfected with HIV-2_(ST) andpRL2P-vpx2CAT, in which anti-Vpx antibody detected 41 and 15 kDaproteins (FIG. 10C). These results demonstrate that Vpr1CAT and Vpx2CATfusion proteins are packaged into virions. However, like in the case ofSN fusion proteins, CAT fusion proteins were also cleaved by the HIVprotease (the Vpx2CAT cleavage product is not visible because ofco-migration migration with the native Vpx protein. CAT cleavageappeared less extensive, based on the intensity of the full-length CATfusion protein on immunoblots.

[0055] Lysates of HIV-1 and HIV-2 viral particles were diluted 1:50 in20 mM Tris-base and analyzed for CAT activity by the method of Allon, etal., Nature 282:864-869 (1979). FIG. 10D indicates that virions whichcontained Vpr1CAT and Vpx2CAT proteins possessed CAT activity. Theseresults show the packaging of active Vpr1- and Vpx2-CAT fusion proteins.

EXAMPLE 14 Virion Incorporated SN and CAT Fusion Proteins areEnzymatically Active

[0056] The ability of Vpr1 and Vpx 2 to deliver functionally activeproteins to the virus particle was further confirmed by sucrose gradientanalysis. Virions derived from HLtat cells co-transfected withHIV-2_(ST) and pLR2P-vpx2 were sedimented in linear gradients of 20-60%sucrose as described above. Fractions were collected and analyzed forviral Gag protein (FIG. 11A) and corresponding CAT activity (FIG. 11B).Peak amounts of Gag protein were detected in fractions 6 and 7 (density1.16 and 1. 17, respectively). Similarly, peak amounts of acetylatedchloramphenicol (Ac-cm) were also detected in fractions 6 and 7.

[0057] Whether virion associated SN fusion protein retained nucleaseactivity was also shown. HIV-1_(SG3) virions containing Vpr1SN wereanalyzed after sedimentation in linear gradients of sucrose (FIG. 11).Since the present invention demonstrated that protease cleavage of SNfusion proteins (FIGS. 7, 8 and 9) markedly reduced Vpr1SN nucleaseactivity (data not shown), these experiments were performed by culturingpSG3/pLR2P-vpr1SN co-transfected cells in the presence of L-689,502 asdescribed above. Immunoblot analysis of sedimented virions revealed peakconcentrations of Gag in fractions 6 and 7 and substantially reduced p55processing (FIG. 11C). Peak SN activity was associated with thefractions that contained the highest concentrations of virus (FIG. 11D).These results thus document that virion incorporation per se does notabrogate the enzymatic activity of Vpr/Vpx fusion proteins, althoughcleavage by the viral protease may inactivate the fusion partners.

[0058] The present invention demonstrated the capability of HIV-1 Vprand HIV-2 Vpx to direct the packaging of foreign proteins into HIVvirions when expressed as heterologous fusion molecules. The transcomplementation experiments with HIV proviral DNA revealed that Vpr1 andVpx2 fusion proteins were also incorporated into replication-competentviruses. Moreover, packaging of the fusion proteins in the presence ofwild-type Vpx and/or Vpr proteins (FIGS. 5, 7 and 8) indicated that theviral signals mediating their packaging were not obstructed by theforeign components of the fusion molecules. Likewise, virion-asociatedSN and CAT fusion proteins remained enzymatically active.

[0059] Based on the immunoblot analysis of VLPs and virions, the presentinvention illustrates that both virion associated CAT and SN/SN* aresusceptible to cleave by the viral protease. There appears to be atleast one cleavage site in CAT and two cleavage sites in the SN/SN*proteins. Based on calculated molecular weights of the major SN/SN*cleavage products, it appears that SN and SN* are cleaved once neartheir C termini and once near the fusion protein junctions. Since thefusion protein junctions of Vpr1SN and Vpx2SN are not identical it isalso possible that these regions differ with respect to theirsusceptibility to the viral protease. Although Vpx2SN/SN* were processedto a lesser extent than Vpr1SN (FIGS. 7 an 8), the major cleavage sitesappear to be conserved. There is no doubt that both the HIV-1 and HIV-2proteases recognize processing sites in the fusion partners and thatthere is sufficient physical contact to enable cleavage. This isevidenced both by the reduction of cleavage product intensities onimmunoblots as well as by an increased enzymatic activity in thepresence of an HIV protease inhibitor.

[0060] The demonstration that Vpr1 and Vpx2 fusion proteins are capableof associating with both VLPs and virions facilitates studies on theseaccessory proteins and on HIV assembly in general. The approach ofgenerating deletion mutants to study protein structure/functionrelationships is often of limited value since this can reduce proteinstability or change the three-dimensional structure of the protein. Inthe case of Vpr, a single amino acid substitution at residue 76 has beenshown to destabilize its expression in infected cells. Studies haveindicated that deletion mutations in vpr and vpx result in prematuredegradation of the proteins following expression. Fusion of Vpr and Vpxmutant proteins with, e.g., SN or CAT as demonstrated by the presentinvention, increase stability.

[0061] The successful packaging of Vpr1/Vpx2SN fusion proteins intovirions indicates their use for accessory protein targeted viralinactivation. The present invention demonstrates that Vpr and Vpx mayserve as vehicles for specific targeting of virus inhibitory molecules,including SN. In contrast to HIV Gag, Vpr and Vpx are small proteinsthat can be manipulated relatively easily without altering virusreplication and thus may represent vehicles with considerableversatility for application to such an antiviral strategy.

[0062] The present invention demonstrated that Vpr and Vpx can serve asvehicles to deliver functionally active enzymes to the HIV virion,including those that may exert an antiviral activity such as SN. Thepresent invention has demonstrated that the concept of accessory proteintargeted virus inactivation is feasible.

[0063] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[0064] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1 10 1 27 DNA HIV-1 YU-2 vpr Primers 1 gccacctttg tcgactgtta aaaaact 272 24 DNA HIV-1 YU-2 vpr Primers 2 gtcctaggca agcttcctgg atgc 24 3 25 DNAHIV-1 HXB2Dgag Primers 3 aaggagagcc atgggtgcga gagcg 25 4 26 DNA HIV-1HXB2Dgag Primers 4 ggggatccct ttattgtgac gagggg 26 5 25 DNA HIV-2ST gagPrimers 5 attgtgggcc atgggcgcga gaaac 25 6 24 DNA HIV-2ST gag Primers 6ggggggcccc tactggtctt ttcc 24 7 28 DNA HIV-1 YU2 vpr Primers 7gaagatctac catggaagcc ccagaaga 28 8 39 DNA HIV-1 YU2 vpr Primers 8cgcggatccg ttaacatcta ctggctccat ttcttgctc 39 9 25 DNA HIV-2ST vpxPrimers 9 gtgcaacacc atggcaggcc ccaga 25 10 39 DNA HIV-2ST vpx Primers10 tgcactgcag gaagatctta gacctggagg gggaggagg 39

What is claimed:
 1. A fusion protein comprising: a first polypeptidesequence corresponding to a portion of a protein functioning inreplication of a virus; and a second polypeptide sequence correspondingto a virus inhibitory protein fragment, said fragment being sufficientto inhibit replication of said virus.
 2. A fusion protein comprising: afirst polypeptide sequence corresponding to a fragment of a viralprotein; and a second polypeptide sequence differing from said fisrtsequence and corresponding to a protein fragment, said fragment coupledto said first sequence.
 3. The fusion protein of claim 2 whereas saidprotein is delivered to a virus.
 4. The fusion protein of claim 2wherein said protein is a Vpr.
 5. The fusion protein of claim 2 whereinsaid portion is a full length amino acid sequence of said protein. 6.The fusion protein of claim 2 wherein said protein is Vpx.
 7. The fusionprotein of claim 3 wherein said virus is selected from the groupconsisting of: virions and virus-like particles, particles containing atleast one viral protein, retrovirus, lentivirus, HIV, and SIV.
 8. Thefusion protein of claim 2 wherein said second polypeptide is selectedfrom the group consisting of: a staphylococcal nuclease, chloramphenicolacetyl transferase, protease, integrase, reverse transcriptase, Vif, Nefand Gag.